The present invention relates to a novel muscurine antagonist obtained from an associated mangrove plant called Salvadora persica Linneaus 1753 collected from estuarine mangrove swamps of Goa, India. Particularly, the present invention relates to the use of methyl palmitate isolated from the plant Salvadora persica as a muscurine antagonist.
Methyl palmitate is a fatty acid methyl ester. It is prepared commercially from naturally occurring edible fats and oils. These compounds are widely used indirectly in a wide range of food, pharmaceutical, cosmetic and industrial applications (Pearson, R., Report of the FAME (Fatty Acid Methyl Esters) Task Force, Jan. 9, 1997). In the same report, data on methyl esters including methyl palmitate, which were tested for oral toxicity in rats, is given. Methyl palmitate was proved to be nontoxic. The report also described that administration of an emulsion of methyl palmitate to mice by oral intubation or intraperitoneal injection produced no alteration in organ weight or phagocytic function of the reticuoendothelial system. In Environment Mutagen 8(Suppl 7):1-119 (1986) (quoted from Pearson, R., supra), it was reported that methyl palmitate was devoid of any mutagenic effect based on the tests on Salmonella and mammalian microsome assays. The methyl palmitate though showed sensitivity of rabbit""s skin to the compound, the results with human skin proved to have a very mild effect. Methyl esters of fatty acids can be used as supplementary source of fat for animal feeds.
xe2x80x9cMuscarinic actionsxe2x80x9d are the actions produced as a result of acetylcholine released from the post-ganglionic parasympathetic nerve endings or the actions resulting from exogenously administered acetylcholine on the receptors of organs with post-ganglionic parasympathetic nerve supply. The muscarinic actions of both endogenously released, as well as exogenously administered, acetylcholine are blocked by atropine. The designation xe2x80x9cmuscarinic actionsxe2x80x9d comes from the fact that these actions are similar to those produced by the poisonous mushroom alkaloid muscarine (R. S. Satoskar and S. D. Bhandarkar, Pharmacology and pharmacotherapeutics, Vol.1., Popular Prakashan, Bombay (1990).
Acetylcholine acts on two types of receptors, namely muscarinic and nicotinic (Goodman and Gilman""s: The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill Health Professions Division, N.Y.; pp. 278-279 (1997)). Since the activity of the crude extract of the Salvadora persica plant was seen on smooth muscle, only muscarinic receptors have been studied (Nazarine, F., Ph.D. Thesis, Goa University, 1998).
A large number of binding and functional studies have indicated the existence of 5 subtypes of muscarinic receptors namely, M1, M2, M3, M4 and M5 (N. J. M. Birdsall and F. C. Hulme, Trends In Pharmacological Sciences, 4:459-461 (1983)). The most important are the M1, M2 and M3 muscarinic receptor subtypes. M1 receptors are found in the stomach and mediate gastric secretions, whereas M2 receptors are found in the myocardium of the heart and M3 are found in the ileum and bladder detrusor muscles and cause smooth muscle contractions.
Thompson et al., in PCT Patent Application No. WO 97/16192, published on May 9, 1997, disclose the antimuscarinic activity of derivatives of piperdin and describe their use in the treatment and/or prevention of myopia, commonly known as near-sightedness.
Haertel et al., in DE Patent No. 4136811 published on May 13, 1993, disclose a skin cleanser especially for removing paint containing methyl, ethyl and/or isopropyl palmitate with some solvents and emulsifiers. Jeanne et al., in U.S. Pat. No. 5,109,022, issued on Apr. 28, 1992, describe a composition which contains methyl palmitate and some other compounds and is useful for repelling flying, biting and stinging insects. U.S. Pat. No. 6,039,950, issued on Mar. 21, 2000, describes formulations of several pharmaceutical grade compounds including fatty esters from the Saw Palmetto, which is a small palm found in the southern United States. U.S. Pat. No. 6,049,013, issued on Apr., 11, 2000, discloses that in methods of producing alcohols, the fatty acids (one example is of methyl palmitate) could be extracted from coconut, palm and palm kernel for this use.
U.S. Pat. No. 5,821,249, which issued on Oct. 13, 1998, describes production of anhydroecgonine methylester (MEG) by heating cocaine base (xe2x80x9ccrackxe2x80x9d) and their use as anticholinergic agents. MEG alone and in combination with cocaine was tested for action on isolated tracheal rings stimulated to contact with acetylcholine.
There is no patent available on the antimuscarinic activity of methyl palmitate. The present invention for the first time discloses the effect of methyl palmitate on M1 receptors by observing gastric acidity by pyloric ligation under basal conditions. Like atropine, the compound methyl palmitate decreased free acidity. However, in the studies conducted, it was 50 times less potent than the standard antagonist atropine. M2 muscarinic receptors found in the conducting tissue and the myocardium of the heart mediate both the bradycardia and the negative inotropic effect of cholinergic stimulation. The present invention relates to the comparative effect of methyl palmitate and atropine on the rate and force of contraction of the guinea pig atria. It describes the inhibitory effect of atropine. The invention describes that the compound methyl palmitate had no such effect in the dosage range used showing that it probably does not block M2 receptors. Though the reason for this effect is not known yet, the present invention for the first time describes the ability of the compound methyl palmitate to distinguish between the muscarinic receptors in the atrium and elsewhere in the body. The very reason that methyl palmitate does not affect the M2 receptors of the atrium confirms the heterogeneity of muscarinic receptors.
As used herein the term xe2x80x9cantimuscarinic activityxe2x80x9d means the antagonistic activity on muscarinic receptors, where xe2x80x9cantagonistic activityxe2x80x9d refers to the inhibitory effect of atropine and the said compound. The xe2x80x9cheterogeneity of muscarinic receptorsxe2x80x9d means the ability of the said compound to distinguish between the muscarinic receptors in the atrium and elsewhere in the body. The xe2x80x9cselectivity of the compoundxe2x80x9d means its inhibitory action only on M1 and M3 muscarinic receptors. The xe2x80x9ccompetitive antagonismxe2x80x9d means the compound produced a shift to the right in the dose response curves of the acetylcholine with the maximum response remaining the same. In gastric acidity experiments, the compound was injected subcutaneously as per the body weight of the rat measured in kilograms and written as Kg. s.c. where xe2x80x9cs.c.xe2x80x9d means subcutaneous.
The approach adopted for antagonistic activity screening by pharmacological methods is described for the first time for this compound. The invention describes that methyl palmitate produces a shift to the right in the dose response curves of acetylcholine with the maximal response remaining the same. It further discloses that the competitive antagonism shown by the said compound is reversible in nature. Methyl palmitate blocks M3 receptors in the same way as atropine, though it is 2.2 times less potent.
The main object of the present invention is to study the compound methyl palmitate purified from the extract of the plant Salvadora persica for its antimuscarinic activity.
Another object of the present invention is to study the heterogeneity of methyl palmitate as a muscarinic receptor.
Still another object of the present invention is to study the comparative effect of methyl palmitate and atropine during in vivo and in vitro experiments.
One more object of the present invention is to detect the effect of methyl palmitate on the dose response curve of acetylcholine.
One other object of the present invention is to detect the effect of methyl palmitate in rats.
Another object of the present invention is to find out the effect of the methyl palmitate in guinea pigs.
The present invention seeks to overcome the drawbacks inherent in the prior art by providing highly efficient and selective methods for screening antimuscarinic activity of a methyl palmitate compound purified from the plant Salvadora persica and discloses the use of this compound as a muscurine antagonist.
Accordingly, the present invention provides a process for obtaining methyl palmitate from a mangrove plant source, namely Salvadora persica, said process comprising:
(i) obtaining an extract from Salvadora persica and
(ii) extracting and purifying the biologically active extract to obtain methyl palmitate from the extract.
Also, the present invention provides a process for preparation of the extract comprising methyl palmitate from the mangrove plant source Salvadora persica, said process comprising:
(i) air-drying the plant parts;
(ii) immersing the plant parts in 90 percent aqueous methanol for one week at room temperature (28xc2x12xc2x0 C.) to obtain a methanolic extract;
(iii) filtering the methanolic extract by conventional methods; and
(iv) evaporating the methalonic extract at room temperature (28xc2x12xc2x0 C.) to obtain a crude extract.
More particularly, the present invention provides a process for the extraction and purification of a biologically active methyl palmitate, useful as a muscarine antagonist, from an extract of Salvadora persica mangrove plant, said process comprising:
a) obtaining a solvent extract from the plant parts of Salvadora persica,
b) testing the extract using methods of pharmacology;
c) fractionating the extract into fractions;
d) testing the fractions using methods of pharmacology;
e) isolating a pure compound by a conventional method;
f) testing the pure compound by using methods of pharmacology; and
g) identifying the compound by conventional methods.
In an embodiment of the present invention, the plant parts of Salvadora persica are selected from leaves, stems and flowers.
In another embodiment of the present invention, the extract is obtained using solvents such as chloroform and hexane.
In yet another embodiment of the present invention, the methyl palmitate molecule is characterized by:
Molecular formula: C16H32O2 
Molecular weight: 256
Melting point: 30xc2x0 C.
As used herein, the term xe2x80x9cextractxe2x80x9d denotes the extract obtained from the plant Salvadora persica. 
In still another embodiment of the present invention, methyl palmitate is capable of distinguishing between muscarinic receptors of atrium and other parts of the body and shows heterogeneity of muscarinic receptors. Methyl palmitate shows competitive antagonism, which is reversible in nature.
In one other embodiment of the present invention, the methyl palmitate obtained inhibits gastric acidity and gastrointestinal disorders. The applicants have found that methyl palmitate inhibits gastric acidity as it exhibits activity on the M1 receptors found in the stomachs of rats. It also exhibits activity against M3 receptors found in the ileal and bladder detrusor muscles of pig.
It has also been found that methyl palmitate does not block M2 receptors and thereby does not have inhibitory effect on the contractions of atrial muscles of guinea pig in the dosage range used.
In still another embodiment of the present invention, methyl palmitate produces a right shift in the dosage response curves of acetylcholine with the maximum response remaining the same.
In one more embodiment of the present invention, methyl palmitate shows muscarinic activity with a milder potency than atropine.
Based on the studies conducted, the invention provides a novel muscurine antagonist obtained from the plant Salvadora persica. The applicants have found that the extract, as well as methyl palmitate obtained from the extract of Salvadora persica, acts as a muscurine antagonist.
Additionally, the invention provides pharmaceutical compositions useful as muscarine antagonists, comprising an effective amount of extract obtained from the mangrove plant Salvadora persica, optionally with conventional additives. The composition may also contain methyl palmitate as the active ingredient. The amount of the active ingredient in the composition may be readily determined by a person skilled in the art depending on factors such as the patient being treated, his body weight, etc. Generally, the effective amount may be in the range of 0.1 to 10 mg/kg body weight.
Further, the applicants have found that the compound methyl palmitate obtained from the plant has the general capacity to impact muscle contractions by slowing down or halting contractions. As a result, it is capable of reducing spasms in smooth muscles, hence acting as an antimuscuranic agent. Thus, the compound acts as an effective xe2x80x9cmuscurine antagonist.xe2x80x9d Preferably, the method of treatment comprises the step of administering a composition containing the compound methyl palmitate obtained from Salvadora persica to a subject in need thereof. The diseases for which the composition may be administered are renal colics, bronchial asthma, abdominal cramps, motion sickness and prevention of premature delivery. The applicants believe that the compound methyl palmitate in the extract is responsible for the above activities.
In an embodiment of the present invention, the dose response curves of the standard agonistic drug acetylcholine were carried out in the absence and presence of methyl palmitate on guinea pig atria for the said activity on M2 and M3 receptors.
In another embodiment of the present invention, for the purpose of comparison, three doses of atropine were used for testing antimuscarinic activity on M3 receptors on ileal and bladder muscles, and the amounts of the doses were:
35xc3x9710xe2x88x926 moles/ml of bath concentration;
86xc3x9710xe2x88x926 moles/ml of bath concentration; and
173xc3x9710xe2x88x926 moles/ml of bath concentration.
In yet another embodiment of the present invention, for comparative purposes, three doses of atropine were administered on M2 receptors and the amounts of the doses were:
0.4xc3x9710xe2x88x926 moles/ml of bath concentration;
0.9xc3x9710xe2x88x926 moles/ml of bath concentration;
1.7xc3x9710xe2x88x926 moles/ml of bath concentration.
In still another embodiment of the present invention, for comparative purposes, three doses of atropine were administered to M1 receptor for testing gastric acidity:
0.01 mg/kg of the body weight of rat;
0.05 mg/kg of the body weight of rat; and
0.10 mg/kg of the body weight of rat.
In another embodiment, the percent inhibition of gastric acidity by the compound methyl palmitate was:
50.72% when the dose was 1 mg/kg of the body weight of rat;
55% when the dose was 3 mg/kg of the body weight of rat; and
91.5% when the dose was 5 mg/kg of the body weight of rat.
Table 1: Effect of methyl palmitate and atropine on gastric acidity in rats.